Identification of Phenylphthalazinones as a New Class of Leishmania infantum Inhibitors

Article abstract of DOI:10.1002/cmdc.201900538

Leishmaniasis is a neglected parasitic disease caused by over 20 different Leishmania species. Current treatments often rely on harsh regimes of pentavalent antimonials such as sodium stibogluconate, while more recent drugs suffer other shortcomings such

Full text of DOI:10.1002/cmdc.201900538

Accepted Article
Title: Identification of Phenylphthalazinones as a New Class of
Leishmania infantum Inhibitors
Authors: Maarten Sijm, Erik de Heuvel, An Matheeussen, Guy Caljon,
Louis Maes, Geert-Jan Sterk, Iwan de Esch, and Rob Leurs
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10.1002/cmdc.201900538
ChemMedChem
Identification of Phenylphthalazinones as a New
Class of Leishmania infantum Inhibitors
Maarten Sijm1, Erik de Heuvel1, An Matheeussen2, Prof. Dr. Guy Caljon2, Prof. Dr. Louis
Maes2, Dr. Geert-Jan Sterk1, Prof. Dr. Iwan J.P. de Esch1 and Prof. Dr. Rob Leurs1*.
1Division of Medicinal Chemistry, Faculty of Sciences, Amsterdam Institute for Molecules,
Medicines and Systems (AIMMS), Vrije Universiteit Amsterdam, De Boelelaan 1108
1081 HZ, Amsterdam, The Netherlands,
2 Laboratory of Microbiology, Parasitology and Hygiene (LMPH), University of Antwerp,
Universiteitsplein 1, 2610, Antwerpen, Belgium.
1
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Abstract
Leishmaniasis is a neglected parasitic disease caused by over 20 different Leishmania species.
Current treatments often rely on harsh regimes of pentavalent antimonials such as sodium
stibogluconate, while more recent drugs suffer other shortcomings such as low stability and rapid
emergence of treatment failure, amongst others. Furthermore, the effectiveness of drugs varies
depending on the infecting Leishmania species, thus there is an urgent need for new and effective
anti-leishmanial drugs. Screening of an in-house compound library identified the
hexahydrophthalazinone NPD-2942 as a low micromolar hit with a pIC50 of 5.8 against L. infantum
and a pIC50 of 4.6 for cytotoxicity against human MRC-5 fibroblasts. To derive Structure-Activity-
Relationships, we modified the cyclohexyl ring of the hexahydrophthalazinone scaffold and 1,2,3-
triazoles were attempted as replacement for the pyrazole ring, amongst other. Ultimately, the 2,3-
pyrazole substituted hexahydrophthalazinone NPD-1289 was identified as the most potent
analogue in this series with a pIC50 of 6.3, although some cytotoxicity towards MRC-5 cells (pIC50
= 5.1) was recorded as well. Replacement of the unsubstituted 2,3-pyrazole with 1,2,3-triazoles
led to compounds with lower anti-leishmanial activity. The current scaffold is a valuable new
starting point for optimization towards novel anti-leishmanial drugs.
2
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Introduction
Leishmaniasis is a neglected parasitic disease which comes in several distinct clinical
manifestations and is caused by over 20 different Leishmania species which are transmitted by
phlebotomine sand flies.[1] Visceral leishmaniasis (VL) is caused by L. donovani and L. infantum,
with 500.000 new infections annually resulting in 50000 deaths, although this number could be
higher due to underreporting.[2] Estimations are that another 1.5 million people are suffering from
cutaneous leishmaniasis (CL), which is caused by almost all other Leishmania species.[3]
For 70 years the standard treatment of leishmaniasis is with pentavalent antimonials meglumine
antimonate (1) and sodium stibogluconate (2).[4] While quite effective outside the Indian
subcontinent, the treatment regimens of 3 daily injections for 30 days, often accompanied with
severe pains, are far from ideal.[3, 5] Furthermore, treatment is relatively expensive and often
requires additional medical supervision as a result of side effects such as vomiting, cardiotoxicity
and hepatotoxicity.[6] Due to different effectiveness of antimonials against the various Leishmania
species and emerging drug resistance, amphotericin B (3) is currently recommended as first-line
treatment.[2-3, 7] Some patients however suffer from infusion reactions like chills and fever, while
serious toxicity has been reported as well.[8] As such, much research has been directed towards
improved formulations of amphotericin B (3) to enhance drug delivery and reduce toxicity.[8]
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ChemMedChem
Figure 1 Pentavalent antimony drugs meglumine antiomonate (1), sodium stibogluconate (2),
amphotericin B (3), miltefosine (4), paromomycin (5) and identified hit NPD-2942 (6).
Due to the high cost and low stability of amphotericin B (3), pentavalent antimonials are still
commonly used in rural areas where a cold chain is absent. Other newly introduced drugs also
have serious concerns. The phospholipid miltefosine (4) is the first oral drug available against
leishmaniasis, initially showing high cure rates of 95% with only minor side effects.[9] However,
more recent reports from the clinic indicate substantial levels of treatment failure.[10] The
aminoglycoside paromomycin (5) shows cure rates up to 85% with a treatment regime for a 35 kg
person costing less than 5 euro.[11] The largest downside of paromomycin is the relatively long
treatment regime of injections for 21 days. Experimental resistance selection also showed a high
4
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10.1002/cmdc.201900538
ChemMedChem
propensity for resistance development when used in monotherapy.[12] Combination treatments with
sodium stibogluconate (2) improve the effectiveness for VL to cure rates of ~93%.[13]
The last decades several drugs have been added to the toolbox against leishmaniasis, although
each drug has its limitations.[14] Furthermore, there is a large interspecies difference of drug
susceptibility and each clinical form of leishmaniasis has different requirements towards the
pharmacokinetics of a drug. Hence, there remains an urgent need for new therapeutics.
To identify new hits against Leishmania, an in-house compound library was screened against
Leishmania infantum. The majority of compounds in this library were previously obtained from
other anti-parasitic programs. From this screening, hexahydrophthalazinone (6) was identified as
a hit with a pIC50 of 5.8 against L. infantum and low toxicity against human MRC-5 cells. To
investigate the Structure-Activity-Relationships (SAR) for this potential new chemical class of
antiparasitic drugs, the cyclohexane part of the hexahydrophthalazinone, the phthalazinone-N-
substituent and the aromatic substituent of the anisole ring were selected for modification.
5
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ChemMedChem
Chemistry
First step of the synthesis of hexahydrophthalazinone analogues was the Friedel-Craft acylation
of 2-bromoanisole with several anhydrides, yielding the corresponding keto-acids (9-10, 12,
scheme 1). The keto-acid precursor (11) for tetrahydrophthalazinone was prepared in two steps;
first a Friedel-Craft acylation was done with maleic anhydride leading to trans-keto-acid 8, which
was followed by a Diels-Alder reaction with 1,3-butadiene resulting in trans-keto-acid 11. Ring-
closure of these keto-acids with hydrazine yielded the desired hexahydrophthalazinone (13),
dihydropyridazinone (14), tetrahydrophthalazinone (15) and phthalazinone (16). An isopropyl
moiety was installed on the unsubstituted phthalazinones and pyridazone using sodium hydride
and isopropylbromide (17-20). In case of tetrahydrophthalazinone (11) the alkylation reaction with
NaH resulted in the conversion of the trans-isomer to the cis-isomer as previously reported by de
Heuvel et al.[15] After the alkylation the final aromatic substituents could be introduced via a
Suzuki cross-coupling resulting in analogues substituted with various pyrazoles (21-23, 26-32), 2-
furan (24) and 3-tiophene (25).
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Scheme 1 Synthesis of various phthalazinones and pyridazinones. Reagents and conditions: a:
AlCl3, anhydride, DCM, rt, 16 h, 58-78 %, b: buta-1,3-diene, THF, 140 oC, 30 min, 10 bar, 97 %.
c: N2H4.H2O, EtOH, rt – 60 oC, 16 h, 20-89 %, d: isopropyl bromide, NaH, DMF, 50 oC, rt, 81-90
o
%, e: R3-B(OH)2 or R3-pinacol, Na2CO3, Pd(dppf)Cl2.DCM, H2O:DME (1:3), 120 C, 1 h, 8-26
%.
Further differentiation of the substituents on the hexahydrophthalazinone nitrogen (scheme 2),
was done by deprotonation with sodium hydride followed by addition of the desired alkylhalides
(33-36). On the alkylated intermediates the so far most promising 4-1H-pyrazolone was installed
using a Suzuki cross-coupling (37-41).
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Scheme 2 Synthesis of hexahydrophthalazinones. Reagents and conditions: a: alkyl bromide or
o
chloride, NaH, DMF, 50 C, rt, 57-92 %, b: pyrazole-B(OH)2, Na2CO3, Pd(dppf)Cl2.DCM,
H2O:DME (1:3), 120 oC, 1 h, 14-29 %.
Introduction of the 1,2,3-triazoles was done starting with isopropyl-hexahydrophthalazinone 18
(scheme 3), which was used in a Sonogashira reaction with trimetylsilylacetylene to yield 42. The
TMS protecting group (42) was removed with potassium hydroxide in MeOH, resulting in the
acetylene substituted building-block (43). The desired azides were prepared in situ by refluxing
sodium azide with the desired alkylhalide in MeOH:H2O (8:1).[16] After 16 hours this mixture was
cooled to room temperature and a portion of the azide solution was added to a mixture of the
acetylene building block (43), CuSO4 and L-ascorbate, yielding the desired 1,2,3-triazoles (44-51)
after stirring overnight at room temperature.
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Scheme 3 Synthesis of triazole substituted hexahydrophthalazinones. Reagents and conditions: a:
a: Pd(PPh3)4, TEA, TMS-acetylene, 100 oC, 1 h, 93 %, b: KOH, MeOH, rt, 86 %, 16 h, c: NaN3,
MeOH:H2O (8:1), 80oC, 16 h, d: R1-N3, CuSO4.5 H2O, L-ascorbate, MeOH: H2O (8:1), rt, 16 h,
34-59 %.
Results and discussion
The first series of analogues synthesized around hit molecule NPD-2942 (6, figure 2) had
modifications at two distinct positions. By modifying R1 and R2, the role of the hexyl ring of the
hexahydrophthalazinone scaffold was investigated, while modifications on R3 were introduced to
investigate the SAR of the hetero-aromatic substituent (table 1). Introduction of a methyl group on
the 1,2-pyrazole (21) reduced antiparasitic activity to 5.1 (pIC50). Instead, installation of a 2,3-
pyrazole ring, either unsubstituted (22) or methylated (23), improved activity compared to the
original hit (6), with both compounds showing pIC50 values of 6.3. Cytotoxicity towards human
MRC-5 cells was however higher for the methylated analogue (23, pIC50 = 5.6, SI = 5) than the
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unsubstituted pyrazole (22, pIC50 = 5.1, SI = 16). Introduction of other heterocyclic rings, such as
a 2-furan (24) and 3-thiophene ring (25) resulted in compounds with decreased activities (pIC50
values around 5.1). The removal of the cyclohexene ring (26-27) resulted in pIC50 values of 4.5,
which is a decrease of 1.3 log units compared to original hit NPD-2942 (6). This shows the
importance of having a cyclohexane ring on this position. Introduction of a cyclohexene ring
instead of a cyclohexane, as in compounds 28-30, resulted in a small increase in activity compared
to the original hit (6) for the unsubstituted 1,2-pyrazole (28) with a pIC50 of 6.0. However, MRC-
5 cytotoxicity of 28 also increased with 0.3 log unit showing 13 fold selectivity towards L. inf. The
unsubstituted 2,3-pyrazole (29) had similar potency as the original hit (6) (pIC50 = 5.7) while the
methylated analogue (30), was more potent with a pIC50 of 6.3. This increased potency of 30 was
however accompanied with an increase of MRC-5 toxicity (pIC50 = 5.4). Replacing the
cyclohexane ring with a phenyl moiety is tolerated, as 2,3-pyrazoles 31 and 32 showed pIC50
values of 5.6 and 6.0 respectively for the unsubstituted and methylated analogues. Both
compounds, however, also showed some MRC-5 cytotoxicity with pIC50 values around 5.0.
After this first round of SAR, it was decided to continue with the unsubstituted 2,3-pyrazole
on the R3 position and a cyclohexene ring on R1 and R2. This compound, NPD-1289 (22), which
has 16 fold selectivity over human cells with a pIC50 of 6.3 and a MRC-5 cytotoxicity of 5.1
(pIC50), showed the best selectivity index in this series. The next position which was investigated
for optimization was the substituent of the phthalazinone nitrogen (R4, Table 2).
Table 1 Phenotypic activity of various N-isopropyl phthalazinones and dihydropyridazinone
against intracellular amastigotes of Leishmania infantum and against MRC-5 cell growth.
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MRC-5
(pIC50) a
Selectivity
Index (SI)b
Cmpnd
R1/R2
A
R3
L. inf (pIC50) a
6
5.8
4.6
16
NPD-2942
21
22
A
A
5.1
6.3
4.6
5.1
3
16
23
24
25
26
A
A
A
B
6.3
5.1
5.2
4.5
5.6
4.5
5
4
< 4.2
4.5
> 10
1
27
28
B
C
4.5
6.0
4.5
4.9
1
13
29
30
31
32
C
C
D
D
5.7
6.3
5.6
6.0
4.6
5.4
5.2
5.0
13
8
3
10
a all reported values are within a standard deviation of ± 0.2 b the SI was calculated by dividing
the activity (IC50) against L. inf by IC50 measured against MRC-5.
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Removal of the isopropyl moiety led to compound 37, which was considered inactive (pIC50 = 4.2)
against L. infantum but did show substantial cytotoxicity towards MRC-5 cells (pIC50 = 5.5).
Installation of a cyclobutylmethyl instead of the isopropyl moiety of 22 resulted in analogue 38.
This molecule shows low micromolar activity against L. infantum (pIC50 = 5.9), but is
accompanied with substantial cytotoxicity (pIC50 = 5.6). The three cyclopropyl-2-oxoethyl 39,
benzyl 40 and 4-pyridinylmethyl 41 analogues all showed even higher cytotoxicity for MRC-5
cells compared to their activities against L. infantum. Based on these findings, further
modifications of the R4 position were discontinued for now.
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Table 2 Phenotypic activity of hexahydrophthalazinones with various N-substituents against
intracellular amastigotes of Leishmania infantum and against MRC-5 cells.
Cmpnd
MRC-5 (pIC50)
Selectivity
Index (SI)b
R4
L. inf (pIC50) a
a
37
38
4.2
5.9
5.5
5.6
0.05
2
39
4.4
5.0
0.25
40
41
5.4
5.4
5.6
6.1
0.63
0.20
a all reported values are within a standard deviation of ±0.2 b the SI was calculated by dividing
the activity (IC50) against L. inf by IC50 measured against MRC-5.
Next, analogues were prepared with modifications on the heteroaromatic ring on R5 (Table 3),
aiming for lower toxicity. Moving away from the pyrazoles, instead 1,2,3-triazoles were
introduced by utilizing click reactions on acetylene substituted hexahydrophthalazinone 43.
Having small aliphatic substituents on the 1,2,3-triazole (44-47) resulted in four compounds with
activities around 5.7 (pIC50), however large differences in MRC-5 toxicity were observed. While
methylcyclopropyl 44 and butyronitrile (47) showed some cytotoxicity against MRC-5 cells (pIC50
= 5.0 and 4.7), methylcyclobutyl (45), cyclopentyl (46) showed virtually no cytotoxicity with pIC50
values of 4.3 and <4.2 respectively, resulting in high SI’s of 25 and >31. Attempts to introduce
13
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some polar side chains, by installing morpholine moieties (48, 49), resulted in compounds with
moderate activities against L. infantum with pIC50 values of 5.1 and 4.7 respectively, while
showing cytotoxicity towards MRC-5 cells at similar concentrations. Installing aromatic moieties,
such as benzyl derivatives 50 and 51, resulted in two compounds with activities around 5.4 (pIC50)
and no observable toxicity against MRC-5 cells (pIC50 < 4.2). While 1,2,3-triazoles showed
substantially lower cytotoxicity than their pyrazole counterparts, their antiparasitic activities are
also lower. The cyclopentyl substituted triazole, NPD-3189 (46) does however show some promise
with a pIC50 of 5.7 and no toxicity.
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Table 3 Phenotypic activity against intracellular amastigotes of Leishmania infantum and against
MRC-5 cell growth by 1,2,3-triazole substituted hexahydrophthalazinones
Cmpnd
L. inf
MRC-5
(pIC50) a
Selectivity
Index (SI)b
R5
(pIC50) a
44
45
5.7
5.7
5.0
4.3
5
25
46
5.7
5.6
< 4.2
4.7
> 31
8
47
48
5.1
4.5
4.7
4.5
3
1
49
50
51
5.2
5.5
< 4.2
< 4.2
> 10
> 20
a all reported values are within a standard deviation of ±0.2 b the SI was calculated by dividing
the activity (IC50) against L. inf by IC50 measured against MRC-5.
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Conclusion
Starting from the screening hit NPD-2942 (6) a series of close analogues has been prepared to
investigate SAR around the hexahydrophthalazinone scaffold. Variations of the core scaffold
shows that removal of the cyclohexane moiety leads to compounds with decreased activity (pIC50s
around 4.5), modification of the cyclohexane to a cyclohexene or phenyl moiety is tolerated,
although this generally does not improve potency. Introduction of several heteroaromatic rings on
R3 revealed that an unsubstituted 2,3-pyrazole on the hexahydrophthalazinone scaffold (22, NPD-
1289) is the most promising analogue of this series with a pIC50 value of 6.3 and a 16-fold
selectivity over toxicity for MRC-5 cells. Close analogue 30 shows a similar potency, however is
less promising with a relative high cytotoxicity (pIC50 value = 5.4, SI = 8). Modifications of the
isopropyl moiety (37-41) led to compounds with high cytotoxicity towards MRC-5 cells and this
position was not further investigated. Replacing the unsubstituted 2,3-pyrazole with 1,2,3-triazoles
(44-51) yielded a set of compounds with lower activity against L. infantum, although higher
selectivity over MRC-5 cells was achieved with methylcyclobutyl 45 (SI = 25) and cyclopentyl 46
(SI >31). Overall, the current series shows the promise of the hexahydrophthalazinone scaffold.
This new chemical class shows submicromolar antileishmanial activity and is a valuable starting
point for further anti-leishmanial hit-to-lead optimization.
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Experimental section biology
Leishmania infantum cellular assay. L. infantum MHOM/MA(BE)/67 amastigotes were collected
from the spleen of an infected donor hamster and used to infect primary peritoneal mouse
macrophages. To determine in vitro antileishmanial activity, 3 × 104 macrophages were seeded in
each well of a 96-well plate. After 2 d of outgrowth, 5 × 105 amastigotes/well were added and
incubated for 2 h at 37 °C. Solutions with or without test compound were subsequently added, and
the plates were further incubated for 5 d at 37 °C and 5% CO2 . Parasite burdens (mean number of
amastigotes/macrophage) were microscopically assessed on 500 cells after Giemsa staining of the
test plates and expressed as a percentage of the blank controls without test compound. In the case
of observed toxicity to the macrophages, the lowest concentration was recorded at which the
toxicity was observed, and this was used as a qualitative phenotypic assessment.[17] As a positive
control Miltefosine was used.
MRC-5 cytotoxicity cellular assay. MRC-5 SV2 cells, originally from a human diploid lung cell line,
were cultivated in MEM, supplemented with L-glutamine (20 mM), 16.5 mM sodium hydrogen carbonate
and 5% FCS. For the assay, 104 cells/well were seeded onto the test plates containing the pre-diluted sample
and incubated at 37°C and 5% CO2 for 72 h. Cell viability was assessed fluorimetrically 4 h after addition
of 10 µg/mL resazurin (excitation 550 nm, emission 590 nm). The results are expressed as percentage
reduction in cell viability compared to untreated controls.[17] Tamoxifen was used as a positive control in
this experiment.
Experimental section chemistry
Chemicals and reagents were obtained from commercial suppliers and were used without further
purification. Anhydrous DMF, THF and DCM were obtained by passing them through an activated alumina
column prior to use. Microwave reactions were executed using a Biotage® Initiator microwave system. 1H
17
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NMR spectra were recorded on a Bruker Avance 250 (250 MHz), Bruker Avance 400 (400 MHz), Bruker
Avance 500 (500 MHz) or Bruker 600 Avance (600 MHz) spectrometer. Data are reported as follows:
chemical shift, integration, multiplicity (s = singlet, d = doublet, dd = double doublet, t = triplet, dt = double
triplet, q = quartet, p = pentet, h = heptet, bs = broad singlet, m = multiplet), and coupling constants (Hz).
Chemical shifts are reported in ppm with the natural abundance of deuterium in the solvent as the internal
reference (CDCl3: δ 7.26, (CD3)2SO: δ 2.50). 13C NMR spectra were recorded on a Bruker Avance 500 (126
MHz) or Bruker Avance 600 (150 MHz). Chemical shifts are reported in ppm with the solvent resonance
resulting from incomplete deuteration as the internal reference (CDCl3: δ 77.16 or (CD3)2SO: δ 39.52).
Systematic names for molecules according to IUPAC rules were generated using the Chemdraw AutoName
program. LC-MS data was gathered using a Shimadzu HPLC/MS workstation with a LC-20AD pump
system, SPD-M20A diode array detection, and a LCMS-2010 EV mass spectrometer. The column used is
an Xbridge C18 5 μm column (100 mm × 4.6 mm). Solvents used were the following: solvent B = ACN,
0.1% formic Acid; solvent A = water, 0.1% formic acid. The analysis was conducted using a flow rate of
1.0 mL/min, start 5% B, linear gradient to 90% B in 4.5 min, then 1.5 min at 90% B, linear gradient to 5%
B in 0.5 min and then 1.5 min at 5% B, total run time of 8 min. All reported compounds have purities >95%,
measured at 254 nm, unless otherwise mentioned. All HRMS spectra were recorded on a Bruker microTOF
mass spectrometer using ESI in positive-ion mode. Column purifications were either carried out
automatically using Biotage equipment or manually, using 60-200 mesh silica. TLC analyses were
performed with Merck F254 alumina silica plates using UV visualization. All reactions were done under
N2 atmosphere, unless specifically mentioned.
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Experimental data
(E)-4-(3-Bromo-4-methoxyphenyl)-4-oxobut-2-enoic acid (8)
To an ice-cooled mixture of 1-bromo-2-methoxybenzene (6)(65 mL, 0.52 mol) and furan-2,5-
dione (77 g, 0.78 mol) in DCM (465 mL) was added AlCl3 (84 g, 0.63 mol). The reaction mixture
was stirred at rt overnight. The orange suspension was quenched in 3 M aq. HCl (1.5 L) and
extracted using EtOAc (4 × 1 L). The combined organic layers were dried over Na2SO4, filtered
and concentrated to obtain a dark yellow solid. Trituration with Et2O provided the product as a
light yellow solid (97.5 g, 66%). 1H NMR (500 MHz, CDCl3): δ 7.89 (d, J = 2.2 Hz, 1H), 7.69 (dd,
J = 8.7, 2.2 Hz, 1H), 7.53 (d, J = 15.4 Hz, 1H), 6.74 (d, J = 8.7 Hz, 1H), 6.51 (d, J = 15.4 Hz, 1H),
3.69 (s, 3H). 13C NMR (126 MHz, DMSO-d6): δ 187.2, 167.1, 166.7, 160.2, 133.8, 133.0, 131.3,
130.6, 112.9, 111.8, 57.3. LC-MS (ESI) m/z found: 285 [M+H]+; retention time: 4.09 minutes.
HRMS (ESI): m/z: [M + H]+ calcd. for C11H10BrO4 284.9757, found 284.9763.
(1R,2S)-2-(3-bromo-4-methoxybenzoyl)cyclohexanecarboxylic acid (9)
cis-hexahydroisobenzofuran-1,3-dione (17.3 g, 112 mmol) was added to a round bottom flask,
followed by 1-bromo-2-methoxybenzene (6) (20 g, 107 mmol) and DCM ( 250 ml). The mixture
was stirred and cooled with an ice-bath. Aluminum trichloride (17.1 g, 128 mmol) was added in
small portions over approximately 5 min, after which the ice-bath was removed and the mixture
stirred for 16 h, by then the mixture was a deep red solution. The mixture was quenched in a
mixture of ice and concentrated hydrochloric acid (3:1) after which a white solid precipitated.
Solids were filtered off, washed with water (3x 50 mL) and dried in vacuo to yield 24 g (63 mmol,
1
58%) of the title compound. H-NMR: (500 MHz, DMSO-d6) δ 12.05 (s, 1H), 8.04 (d, 8.6 Hz,
1H), 7.94 (d, 8.6 Hz, 1H), 7.21 (d, J = 8.6 Hz, 1H), 3.93 (s, 3H), 3.39 (s, 1H), 2.73 – 2.58 (m, 1H),
2.03 (q, J = 9.5 Hz, 1H), 1.93 – 1.78 (m, 2H), 1.78 – 1.67 (m, 1H), 1.67 – 1.56 (m, 1H), 1.44 –
1.23 (m, 2H), 1.24 – 1.10 (m, 1H). LC-MS (ESI) m/z found: 341 [M+H]+; retention time: 4.43
minutes, purity: 85%. HRMS-ESI [M+H]+ calculated for C15H18BrO4: 341.0383, found 341.0392.
trans-6-(3-Bromo-4-methoxybenzoyl)cyclohex-3-ene-1-carboxylic acid (11)
A mixture of keto-acid 8 (70.0 g, 221 mmol) and buta-1,3-diene in THF (~ 13% w/w, 150 mL,
300 mmol) was divided over 8 microwave vials. Each vial was stirred under microwave irradiation
at 140 °C for 30 min (the internal pressure reached ~ 10 bar). The reaction mixtures were pooled
and concentrated in vacuo to obtain the crude product. Trituration with toluene provided the
product as a white solid (72.4 g, 97%). 1H NMR (500 MHz, DMSO-d6): δ 12.30 (s, 1H), 8.16 (s,
1H), 8.07 (d, J = 8.7 Hz, 1H), 7.23 (d, J = 8.5 Hz, 1H), 5.79 – 5.66 (m, 2H), 3.95 (s, 3H), 3.83 –
3.72 (m, 1H), 2.92 – 2.75 (m, 1H), 2.54 – 2.10 (m, 3H), 1.93 – 1.79 (m, 1H). 13C NMR (126 MHz,
DMSO-d6): δ 200.8, 176.4, 159.61 133.4, 130.6, 130.3, 125.8, 125.7, 112.8, 111.6, 57.2, 42.2,
41.6, 29.4, 28.4. LC-MS (ESI) m/z found: 339 [M+H]+; retention time: 4.53 minutes. HRMS (ESI):
m/z: [M + H]+ calcd. for C15H16BrO4 339.0226, found 339.0213.
(4aS,8aR)-4-(3-bromo-4-methoxyphenyl)-4a,5,6,7,8,8a-hexahydrophthalazin-1(2H)-one (13)
19
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Keto-acid 10 (13.0g, 38.1 mmol) was charged to a round bottom flask followed by ethanol (150
mL). This suspension was stirred and hydrazine hydrate (50-60% (20 mL, 381 mmol) was added
in portions. The mixture was heated at 60oC for 16 h after which it was allowed the cool to rt while
stirring. White precipitation was visible and 50 mL of water was added, increasing precipitation.
Solids were filtered off and dried in vacuo to yield 11.4 gram (33.8 mmol, 89%) of the title
compound as a white solid. 1H-NMR: (500 MHz, DMSO-d6) δ 10.89 (s, 1H), 7.97 (d, J = 2.1 Hz,
1H), 7.76 (dd, J = 8.7, 2.2 Hz, 1H), 7.15 (d, J = 8.8 Hz, 1H), 3.88 (s, 3H), 3.29 – 3.20 (m, 1H),
2.71 (s, 1H), 2.28 (d, J = 12.5 Hz, 1H), 1.67 (d, J = 12.6 Hz, 1H), 1.56 – 1.47 (m, 2H), 1.45 – 1.33
(m, 2H), 1.30 – 1.16 (m, 2H). LC-MS (ESI) m/z found: 337 [M+H]+; retention time: 4.41 minutes.
HRMS-ESI [M+H]+ calculated for C15H18BrN2O2: 337.0546, found 337.0536.
(4aS,8aR)-4-(3-bromo-4-methoxyphenyl)-2-isopropyl-4a,5,6,7,8,8a-hexahydrophthalazin-1(2H)-
one (17)
Phthalazinone 13 (5.0 g, 14.8 mmol) was dissolved in DMF (100 mL) and sodium hydride (60%
in mineral oil) (652 mg, 16.3 mmol) was added. The mixture was stirred for 15 min after which 2-
bromopropane (1.67 mL, 17.8 mmol) was added and the mixture was stirred for 16 h at 60oC. The
reaction was quenched pouring the mixture in water (250 mL) and the aqueous layer was extracted
with MTBE (250 mL). The organic layer was washed with sat. aqueous NaHCO3 (2x 250 mL) and
brine (250 mL) after which the organic layer was dried over Na2SO4. The solids were filtered off
and volatiles were evaporated in vacuo after which the remaining crude was purified over SiO2
using a gradient of 10% EtOAc in heptane towards 50% EtOAc in heptane yielding 4.6g (12.1
mmol, 82%) of the title compound as a white solid. 1H-NMR: (500 MHz, CDCl3) δ 7.98 (s, 1H),
7.69 (d, J = 8.6 Hz, 1H), 6.90 (d, 1H), 5.03 (hept, J = 6.4 Hz, 1H), 3.03 – 2.93 (m, 1H), 2.63 (s,
1H), 2.60 – 2.47 (m, 1H), 1.84 – 1.72 (m, 1H), 1.68 – 1.56 (m, 2H), 1.40 – 1.30 (m, 3H), 1.29 (d,
J = 6.6 Hz, 3H), 1.21 (d, J = 6.7 Hz, 3H). 13C-NMR: (126 MHz, CDCl3) δ 166.45, 156.68, 151.24,
130.75, 129.19, 126.15, 112.06, 111.47, 56.36, 46.40, 36.69, 35.48, 26.90, 25.67, 24.28, 23.98,
20.65, 20.18. LC-MS (ESI) m/z found: 379 [M+H]+; retention time: 5.51 minutes. HRMS-ESI
[M+H]+ calculated for C18H24BrN2O2: 379.1016, found 379.1009.
2-isopropyl-4-(4-methoxy-3-(1H-pyrazol-4-yl)phenyl)-4a,5,6,7,8,8a-hexahydrophthalazin-
1(2H)-one (22)
Phthalazinone 17 (100 mg, 0.26 mmol) and (1H-pyrazol-4-yl)boronic acid (35 mg, 0.32 mmol)
were added to a microwave tube with a stirring bean, subsequently DME (3 ml) and 1M sodium
carbonate (0.9 mL, 0.9 mmol) were added and the mixture was degassed for 5 minutes with N2.
After addition of PdCl2(dppf).CH2Cl2 (20 mg, 0.026 mmol) the mixture was degassed for another
2 min and the vessel was sealed and heated for 1 h at 120oC. The mixture was diluted with EtOAc
(25 mL), filtered over Celite and the organic layer was washed with water (2x 25 mL) and brine
(25 mL). The organic layer was dried over Na2SO4, solids were filtered off and the residue was
concentrated in vacuo. Remaining crude was purified over SiO2 using a gradient of 50% heptanes
in EtOAc towards 5% MeOH in EtOAc to yield the 18 mg (0.05 mmol, 19%) of title compound
as an off-white solid. 1H-NMR: (500 MHz, CDCl3) δ 8.08 (s, 2H), 8.04 (d, J = 2.3 Hz, 1H), 7.64
(dd, J = 8.6, 2.3 Hz, 1H), 6.99 (d, J = 8.6 Hz, 1H), 5.08 (hept, J = 6.6 Hz, 1H), 3.95 (s, 3H), 3.13
– 3.03 (m, 1H), 2.72 – 2.65 (m, 1H), 2.61 – 2.52 (m, 1H), 1.85 – 1.76 (m, 1H), 1.73 – 1.60 (m,
2H), 1.49 – 1.35 (m, 4H), 1.33 (d, J = 6.6 Hz, 3H), 1.25 (d, J = 6.6 Hz, 3H). 13C-NMR: (126 MHz,
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CDCl3) δ 166.60, 156.95, 152.67, 133.16, 127.93, 125.30, 125.22, 121.55, 117.70, 110.93, 55.65,
46.40, 36.76, 35.61, 25.76, 24.44, 24.06, 22.06, 20.69, 20.24. LC-MS (ESI) m/z found: 367
[M+H]+; retention time: 4.55 minutes. HRMS-ESI [M+H]+ calculated for C21H27N4O2: 367.2129,
found 367.2116.
(4aS,8aR)-4-(3-bromo-4-methoxyphenyl)-2-(cyclobutylmethyl)-4a,5,6,7,8,8a-
hexahydrophthalazin-1(2H)-one (33)
Phthalazinone 13 (450 mg, 1.33 mmol) was dissolved in DMF (10 mL) and sodium hydride (60%
in mineral oil) (59 mg, 1.5 mmol) was added. The mixture was stirred for 15 min after which
(bromomethyl)cyclobutane (0.18 mL, 1.6 mmol) was added and the mixture was stirred for another
16 h. The reaction was quenched pouring the mixture in water (50 mL) and the aqueous layer was
extracted with MTBE (50 mL). The organic layer was washed with sat. aqueous NaHCO3 (2x 50
mL) and brine (50 mL) after which the organic layer was dried over Na2SO4. The solids were
filtered off and volatiles were evaporated in vacuo after which the remaining crude was purified
over SiO2 using a gradient of 10% EtOAc in heptane towards 70% EtOAc in heptane yielding 430
mg (1.06 mmol, 79%) of the title compound as a white solid. 1H-NMR: (500 MHz, CDCl3) δ 7.97
(d, J = 2.1 Hz, 1H), 7.66 (dd, J = 8.6, 2.1 Hz, 1H), 6.90 (d, J = 8.7 Hz, 1H), 4.13 (dd, J = 13.2, 7.7
Hz, 1H), 3.93 (s, 3H), 3.65 (dd, J = 13.2, 7.1 Hz, 1H), 3.05 – 2.95 (m, 1H), 2.75 (h, J = 7.8 Hz,
1H), 2.70 – 2.64 (m, 1H), 2.59 – 2.49 (m, 1H), 2.07 – 1.97 (m, 2H), 1.93 – 1.76 (m, 5H), 1.69 –
1.59 (m, 3H), 1.45 – 1.23 (m, 5H). 13C-NMR: (126 MHz, CDCl3) δ 167.17, 156.74, 151.27, 130.83,
128.89, 126.20, 112.11, 111.47, 56.37, 53.30, 36.40, 35.96, 26.12, 26.09, 25.71, 24.40, 23.95,
22.00, 18.46. LC-MS (ESI) m/z found: 405 [M+H]+; retention time: 5.91 minutes. HRMS-ESI
[M+H]+ calculated for C20H26BrN2O2: 405.1172, found 405.1165.
(4aS,8aR)-2-(cyclobutylmethyl)-4-(4-methoxy-3-(1H-pyrazol-4-yl)phenyl)-4a,5,6,7,8,8a-
hexahydrophthalazin-1(2H)-one (38)
Phthalazinone 33 (150 mg, 0.37 mmol) and (1H-pyrazol-4-yl)boronic acid (62 mg, 0.56 mmol)
were added to a microwave tube with a stirring bean, subsequently DME (4 ml) and 1M sodium
carbonate (2 mL, 2.0 mmol) were added and the mixture was degassed for 5 min with N2. After
addition of PdCl2(dppf).CH2Cl2 (30 mg, 0.037 mmol) the mixture was degassed for another 2
minutes and the vessel was sealed and heated for 1 h at 120oC. The mixture was diluted with
EtOAc (25 mL), filtered over Celite and the organic layer was washed with water (2x 25 mL) and
brine (25 mL). The organic layer was dried over Na2SO4, solids were filtered off and the residue
was concentrated in vacuo. Remaining crude was purified over SiO2 using a gradient of 50%
heptanes in EtOAc towards 5% MeOH in EtOAc to yield 37 mg (0.09 mmol, 25%) of the title
compound as an off-white solid. 1H-NMR: (500 MHz, CDCl3) δ 8.09 (s, 2H), 8.00 (d, J = 1.8 Hz,
1H), 7.59 (dd, J = 8.6, 2.0 Hz, 1H), 6.97 (d, J = 8.7 Hz, 1H), 4.17 (dd, J = 13.2, 7.6 Hz, 1H), 3.94
(s, 3H), 3.68 (dd, J = 13.2, 7.1 Hz, 1H), 3.14 – 3.03 (m, 1H), 2.82 – 2.68 (m, 2H), 2.61 – 2.51 (m,
13
1H), 2.12 – 1.97 (m, 2H), 1.94 – 1.76 (m, 5H), 1.73 – 1.59 (m, 2H), 1.50 – 1.29 (m, 4H). C-
NMR: (126 MHz, CDCl3) δ 167.35, 157.02, 152.87, 133.38, 127.59, 125.35, 125.22, 121.77,
117.58, 110.94, 105.69, 55.63, 53.25, 36.47, 36.12, 34.86, 26.20, 25.78, 24.55, 24.02, 22.08, 18.52.
LC-MS (ESI) m/z found: 393 [M+H]+; retention time: 4.83 minutes. HRMS-ESI [M+H]+
calculated for C23H28N4O2: 393.2285, found 393.2276.
21
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(4aS,8aR)-2-isopropyl-4-(4-methoxy-3-((trimethylsilyl)ethynyl)phenyl)-4a,5,6,7,8,8a-
hexahydrophthalazin-1(2H)-one (42)
Phthalazinone 13 (2.0 g, 5.3 mmol) was added to a microwave tube and TEA (12 ml) was added.
The mixture was degassed with N2 for 5 min after which Tetrakis (0.61 g, 0.53 mmol) and CuI
(0.1 g, 0.53 mmol) were added. The tube was heated in the microwave at 100 °C for 1 h, after
which the mixture was allowed to cool to rt, diluted with MTBE (25 mL) and filtered over Celite.
The organic layer was washed with saturated aqueous NaHCO3 (2x 25 mL) and brine (25 mL),
after which it was dried over Na2SO4 and solids were filtered off. The remaining black crude was
concentrated in vacuo and purified over SiO2 using a gradient of heptane towards 25% EtOAc in
1
heptane yielding 1.95 g (4.9 mmol, 93%) of the title compound as a white solid. H-NMR: (500
MHz, CDCl3) δ 7.83 – 7.76 (m, 2H), 6.89 (d, J = 8.7 Hz, 1H), 5.05 (hept, J = 6.6 Hz, 1H), 3.92 (s,
3H), 3.07 – 2.99 (m, 1H), 2.63 (s, 1H), 2.55 (d, J = 9.8 Hz, 1H), 1.83 – 1.75 (m, 1H), 1.68 – 1.60
(m, 2H), 1.58 (s, 2H), 1.41 – 1.32 (m, 2H), 1.31 (d, J = 6.6 Hz, 3H), 1.23 (d, J = 6.7 Hz, 3H), 0.29
(s, 8H). LC-MS (ESI) m/z found: 397 [M+H]+; retention time: 6.09 minutes.
(4aS,8aR)-4-(3-ethynyl-4-methoxyphenyl)-2-isopropyl-4a,5,6,7,8,8a-hexahydrophthalazin-
1(2H)-one (43)
Phthalazinone 42 (2.0 g, 5.0 mmol) was charged to a round bottom flask, followed by MeOH (50
mL) and 2.5M aqueous potassium hydroxide. The mixture was stirred overnight at rt for 16 h, after
which the aqueous layer was extracted with EtOAc (50 mL). The organic layer was washed with
brine (50 mL) and dried over Na2SO4 after which the solids were filtered off. Volatiles were
removed in vacuo, after which the remaining crude was purified over SiO2 using a gradient of
10% EtOAc in heptanes towards 70% EtOAc in heptanes yielding 1.4 g (5.0 mmol, 86%) of the
title compound as a white solid. 1H-NMR: (500 MHz, CDCl3) δ 7.87 (d, J = 2.3 Hz, 1H), 7.81 (dd,
J = 8.8, 2.3 Hz, 1H), 6.93 (d, J = 8.8 Hz, 1H), 5.05 (hept, J = 6.6 Hz, 1H), 3.95 (s, 3H), 3.35 (s,
1H), 3.07 – 2.97 (m, 1H), 2.67 – 2.62 (m, 1H), 2.59 – 2.50 (m, 1H), 1.84 – 1.76 (m, 1H), 1.68 –
1.60 (m, 2H), 1.47 – 1.32 (m, 4H), 1.30 (d, J = 6.6 Hz, 3H), 1.23 (d, J = 6.7 Hz, 3H). 13C-NMR:
(126 MHz, CDCl3) δ 166.47, 161.36, 151.60, 131.59, 128.01, 127.84, 111.30, 110.66, 81.51,
79.74, 56.11, 46.36, 36.70, 35.47, 25.69, 24.28, 24.01, 22.00, 20.65, 20.18. LC-MS (ESI) m/z
found: 325 [M+H]+; retention time: 5.12 minutes. HRMS-ESI [M+H]+ calculated for C20H25N2O2:
325.1911, found 325.1904.
(4aS,8aR)-4-(3-(1-(cyclopropylmethyl)-1H-1,2,3-triazol-4-yl)-4-methoxyphenyl)-2-isopropyl-
4a,5,6,7,8,8a-hexahydrophthalazin-1(2H)-one (44)
(azidomethyl)cyclopropane was freshly prepared by refluxing (bromomethyl)cyclopropane (0.49
g, 3.6 mmol) and sodium azide (0.47 g, 7,2 mmol) in 4 mL MeOH and 0.5 mL H2O for 16 h. To a
tube 1 mL of the azide solution (0.8 M) was added followed by alkyne 43 (50 mg, 0.15 mmol),
(+)-sodium L-ascorbate (3 mg, 0.015 mmol) and CuSO4.5 H2O (4 mg, 0.015 mmol), after which
the tube was sealed. The reaction was stirred overnight at room temperature after which the
reaction was quenched in 1M aqueous Na2CO3 (25 mL) and extracted with EtOAc (2x 25 mL).
The organic layer was washed with 1M aqueous Na2CO3 (2x 25 mL) and brine (25 mL), dried
over Na2SO4 and filtered off. The remaining residue was concentrated in vacuo and purified over
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SiO2 using a gradient of 70% heptanes in EtOAc towards 30% heptanes in EtOAc yielding 27 mg
(0.06 mmol, 43 %) of the title compound as a white solid. 1H-NMR: (500 MHz, CDCl3) δ 8.66 (d,
J = 2.3 Hz, 1H), 8.13 (s, 1H), 7.92 (dd, J = 8.8, 2.4 Hz, 1H), 7.03 (d, J = 8.8 Hz, 1H), 5.06 (hept,
J = 6.7 Hz, 1H), 4.28 (d, J = 7.2 Hz, 2H), 3.99 (s, 3H), 3.28 – 3.18 (m, 1H), 2.70 – 2.64 (m, 1H),
2.59 – 2.50 (m, 1H), 1.81 – 1.57 (m, 5H), 1.49 – 1.33 (m, 5H), 1.32 (d, J = 6.6 Hz, 3H), 1.25 (d, J
= 6.7 Hz, 3H), 0.75 – 0.68 (m, 2H), 0.52 – 0.44 (m, 2H). 13C-NMR: (126 MHz, CDCl3) δ 166.70,
156.47, 152.97, 142.69, 138.93, 133.38, 128.32, 126.80, 125.27, 122.79, 121.77, 119.38, 111.22,
55.70, 54.95, 46.34, 36.80, 35.38, 25.64, 24.27, 24.01, 22.07, 20.66, 20.21, 11.31, 4.24. LC-MS
(ESI) m/z found: 422 [M+H]+; retention time: 5.12 minutes. HRMS-ESI [M+H]+ calculated for
C24H31N5O2: 422.2551, found 422.2543.
Corresponding author
Rob Leurs, r.leurs@vu.nl
Author contributions
M.S. and E. d. H. contributed to the synthesis of the molecules, A.M., G.C. and L.M. were
involved with the parasitological screenings, M.S., L.M., G.S and R.L. were involved in the design
of the molecules and experiments. L.M, G.C., G.S, I.J.P de E and R.L obtained the necessary
funding. M.S., G.S. and R.L. contributed to the writing of this manuscript. All authors have read
and checked the manuscript.
Funding Sources
The PDE4NPD project was funded by the European Union under the FP-7-Health program,
project ID: 602666.
Acknowledgements
Authors would like to thank H. Custers and A. van der Stolpe for their technical assistance.
List of abbreviations
ACN: acetonitrile, bs: broad singlet, CL: cutaneous leishmaniasis, cLogP: calculated logarithm
of the partition-coefficient, d: doublet, DCM: dichloromethane, dd: double doublet, DME:
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dimethoxyethane, dt: double triplet, DMF: dimethylformamide, dppf: 1,1′-Ferrocenediyl-
bis(diphenylphosphine), ESI: electron spray ionization, EtOAc: ethylacetate, EtOH: ethanol, h:
heptet, h: hour, HPLC: high pressure liquid chromatography, HRMS: high resolution mass
spectroscopy, Hz: hertz, J: coupling constant, L. inf.: Leishmania infantum, LC-MS: liquid
chromatography- mass spectroscopy, LDA: lithium di-isopropyl amide, m: multiplet, MeOH:
methanol, MHz: megahertz, MRC-5: medical research council cell strain 5, MS: mass
spectroscopy, MTBE: methyl tert-butyl ether, nm = nanometer, NMR: nuclear magnetic
resonance, p = pentet, pIC50 = inverse logarithm of the concentration at which 50% is inhibited,
ppm = parts per million, q = quartet, s: singlet, SAR: structure activity relationship, SEM: standard
error of the mean, SI: selectivity index, t: triplet, THF: tetrahydrofuran, VL: visceral leishmaniasis.
Table of contents
Leishmaniasis is caused by protozoan parasites of the Leishmania family and targets millions of
people worldwide. Currently used drugs have several limitations and/or side effects. As such the
need for new molecules in the drug discovery pipeline is high. Phenylphthalazinone NPD-2942
was identified as a hit and 25 close analogues were prepared to investigate SAR. Several promising
analogues were identified which are useful starting points for further optimization.
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10.1002/cmdc.201900538
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